Methods for purification of bacterial cells and components

ABSTRACT

The present invention relates to a method for the selective purification of bacterial cells and/or cell components, whereby the purification is performed by means of a solid support.

[0001] The present invention relates to a method for the selectivepurification of bacterial cells and/or cell components wherein thepurification is performed by means of a solid support.

[0002] The starting point of almost any further processing, analysis, orisolation of cell components is the enrichment of the cells, named cellharvest, usually being carried out by means of centrifugation. Thiscentrifugation step is the main problem of the entire automation ofmethods, for example of the plasmid purification, since in addition tothe high technical complexity for the integration of a centrifuge in arespective processing robot an extremely high precision of the start andstop position of the centrifugation process is required. Automaticmethods of the further processing analysis or isolation of cellcomponents usually starts with cells being enriched, centrifuged, orsedimented outside of the processing robot. For example, a nearly entireautomation of the relevant methods is essential for a rapid analysis ofcomplete genomes, proteoms, and also for a rapid determination of thestructure and function in high throughput methods. The automation forexample in the genome analysis has already been highly advanced: Thebacterial growth as well as the plasmid isolation may be carried outautomatically. However, an entire automation of the methods includingthe cell harvest is still not feasible. Particularly a selective cellharvest, that is the specific enrichment of particular cells out of acell mixture, is not possible with the presently used methods.

[0003] The cell harvest is usually carried out with the followingmethods: The standard method of the cell harvest is the unspecificcentrifugation of the bacterial cultures. A microplate centrifuge isnecessary especially in those methods which are constructed for a higherthroughput. However, the centrifugation as such is not suitable for anautomation.

[0004] Though the filtration of the cultivated cells with respectivefilter membranes is feasible, that filtration also allows only anunspecific enrichment of the cells. In addition, the method is highlyaccident sensitive with respect to plugging in highly enriched cellsuspensions and the high viscosity of the solutions as a consequencethereof.

[0005] The fluorescence activated cell sorting is a method in which avery thin liquid thread is used allowing the sorting and enrichment ofsingle fluorescence labelled cells by means of the laser. By use of arespective fluorescence label, a certain specificity of the enrichmentis possible, but due to the thin liquid thread, the method is limited tosmall volumes and thus to a low throughput. Therefore, only small cellamounts may be enriched, which is not sufficient for a furtherprocessing or an analysis of cell components. The high costs for theapparative equipment also prevent a strong propagation of the techniqueand slow down the simultaneous work necessary for a high throughput cellharvest.

[0006] The cells are bound directly to magnetic particles via ionicinteractions and are locally concentrated by applying a magnetic fieldin case of the magnetic cell separation technique. Those methods for theunspecific concentration of bacterial cells have been distributedrecently for the entire automation of the processing of plasmid orgenomic DNA including the cell harvest by Chemagen (EP 1 118 676),Genpoint (WO 01/53525, WO 98/51693), Merck (WO 00/29562), as well asPromega (U.S. Pat. No. 6,284,470) or Amersham (WO 91/12079). However,these magnetic particles exhibit the drawback that on the one hand theybind the bacterial cells in an unspecific manner, and on the other handthey do not bind every bacterial species equally well. Due to theunspecificity of the binding, even different binding efficiencies invarious strains of a species have been observed (Merck WO 00/29562).

[0007] Thus, one object of the present invention is the provision of amethod which is feasible to selectively and fully automatically enrichedbacterial cells and cell components and which may be incorporated intoan automated analysis or isolation method. A further object of thepresent invention is the provision of solid supports for the selectiveenrichment of bacterial cells or cell components.

[0008] The object is achieved by the subject matter defined in theclaims.

[0009] The following figures illustrate the invention.

[0010]FIG. 1 displays in a graphic the time dependency of thep12-dependent binding of E. coli to magnetic beads. The values indicatethe β-galactosidase activity of immobilised cells in relative units. VIK(rhombus symbols) stands for: two-step method (preincubation of cellsand p12, subsequent binding to magnetic beads). VC (squares) stands for:one-step method (precoating of p12 to magnetic beads, subsequentimmobilising of cells). −p12 (triangles) stands for: background(unspecific cell binding to beads without p12).

[0011]FIG. 2 displays in a graphic the binding of E. coli to magneticbeads via the bacteriophage T4. The values indicate the β-galactosidaseactivity of immobilised cells in relative units. VIK stands for:two-step method (preincubation of cells and T4, subsequent binding tomagnetic beads). VC stands for: one-step method (precoating of T4 tomagnetic beads, subsequent immobilising of cells). K stands for:background (unspecific cell binding to beads without T4).

[0012]FIG. 3 displays in a graphic comparison the yield of E. coli cellsfrom different media. −p12 denotes the results without N-strep-p12. +p12denotes the results with N-strep-p12. Hatched bars show the results withthe strain E. coli LE392, filled in bars denote the results with thestrain E. coli JM83. LB, SOB, SOC, TB, and YT 2× denote the respectivemedia being used in the experiment. The values are shown as yield in %of the used cells, determined over the scattering of the supernatant at600 nm after pelleting of the bound cells by means of a magnet.

[0013]FIG. 4 shows graphically the result of the plasmid isolation afterharvest of E. coli with p12 according to the two-step method. StrainDH10b with plasmid pUC19. 1: centrifuged cells, DNA isolation via solidphase extraction, 2: cells harvested with the two-step method accordingto the present invention, DNA isolation via solid phase extraction, 3:centrifuged cells, DNA isolation via magnetic beads, 4: cells harvestedwith the two-step method according to the present invention, DNAisolation via magnetic beads, 5: standard.

[0014]FIG. 5 shows in a graphic display the enrichment of E. coli cellsfrom 10 ml culture volume, starting from a cell suspension of differentcell densities (10⁹-10⁷CFU/ml). Graph A: the filled in bars indicate theβ-galactosidase activity of the cells bound via N-strep-p12, the hatchedbars indicate the background without p12. Graph B: the filled in barsindicate the β-galactosidase activity of cells bound via T4-bio, thehatched bars indicate the background without T4.

[0015]FIG. 6 shows schematically the harvest of living E. coli viabiotinylated p12 and streptavidin beads. The filled in bars indicate thescattering at 600 nm after two hours of growth. The hatched barsindicate the β-galactosidase activity of the cells. The abbreviationsstand for: P12: the determinations of the cells immobilised to thebeads, P12-EDTA: determinations of the cells stripped from the beadsafter the binding (supernatant after EDTA treatment), EDTA-p12:immobilising of the cells to the beads was prevented by the presence ofEDTA; determinations of the unspecific cells to the beads, K: controlexperiment without p12; determinations at the beads.

[0016]FIG. 7 shows in a table the selective binding of p12 to bacterialcells. Table A lists the p12-dependent binding of different E. colistrains, table B shows the specificity of the p12-dependent binding. Theabbreviation n.d. indicates not determined, +indicates the binding ofp12 to the named bacteria, −indicates no binding of p12 to the cells.

[0017]FIG. 8 shows graphically the specificity of the p12-dependentbinding. The values indicate the yield of immobilised cells, determinedover the scattering in the supernatant at 600 nm. The hatched barsindicate the values for the cell binding over N-strep-p12 (=specificbinding). The dark filled in bars show the control without p12(=unspecific adsorption). The light filled in bars indicate the valuesfor the cell binding with p12, in the presence of 10 mM EDTA(=unspecific adsorption), however.

[0018]FIG. 9 shows images of light microscopy and fluorescencemicroscopy of the selective binding of E. coli to magnetic beads viaT4-bio in a mixed culture of E. coli and Serratia marrescens. The E.coli cells were fluorescence labelled with FITC-labelled T4p12. Theimages on the left show exposures of light microscopy, the images on theright show exposures of fluorescence microscopy. The upper images showexposures of an experiment without T4-bio, the lower images showexposures of an experiment with T4-bio.

[0019]FIG. 10 shows graphically the result of the cell harvest after thetwo-step method with N-strep-p12 in solutions with various cell numbers.S/N-ratio indicates the signal to noise-ratio (signal with N-strep-p12divided by the signal without N-strep-p12) of the β-galactosidasereaction of the bound E. coli cells, CFU/ml indicates the used cells(cell forming units) per ml. The squares indicate the results of theβ-galactosidase activity of the measurement with a luminescentsubstrate. The rhombi indicate the results of the β-galactosidaseactivity of the measurement with a fluorescent substrate.

[0020]FIG. 11 shows graphically the result of the harvest of E. colicells after the one-step method with T4p12 which is covalently bound tomagnetic EM2-100/49 beads (Merck Eurolab). +p12 indicates the resultswith beads with T4p12, −p12 indicates the results with beads withoutT4p12, DSMZ 613 indicates the results with the strain E. coli DSMZ 613,DSMZ 13127 indicates the results with the strain E. coli DSMZ 13127.

[0021]FIG. 12 shows graphically the results of the harvest of E. colicells after the one-step method with T4p12 adsorbed to magneticPVA-beads. The rhombi indicate the results with the beads PVA-011(Chemagen), the squares indicate the results with the beads PVA-012. Thecontinuous lines indicate the results with beads having adsorbed T4p12,dashed lines indicate beads without T4p12. The OD600 values indicate the%-values of harvested cells in % of the scattering of the supernatantafter pelleting of the bound cells by means of a magnet.

[0022] The term “phage proteins” or “bacteriophage proteins”, as usedherein, refers to all bacteriophage proteins participating in therecognition and binding of the bacterial cells or the cell components.Said proteins my be localised depending on the morphological property ofthe phages, for example directly on the phage coat or on specificrecognition structures, namely the tail fibres. Thus, the term“bacteriophage tail proteins” refers to phage proteins displaying thebacteriophage tail or being a part of the bacteriophage tail.

[0023] The term “specificity” as used herein means that thebacteriophages or phage proteins recognise and bind only a single genusor species, or a sub-species of bacterial cells or cell components, aswell as that some bacteriophages or phage proteins recognise and bindspecific bacteria groups.

[0024] The term “enrichment” or “purification” as used herein means thespecific separation of bacterial cells or cell components from theaqueous solution, for example from the culture medium, in which thebacterial cells or cell components are located. The purification orenrichment is carried out by means of solid supports, for examplemagnetic particles, glass particles, agarose particles, reaction tubes,or microtiter plates.

[0025] One aspect of the present invention refers to the provision ofmethods for selective purification of bacterial cells or cellcomponents, comprising the following steps: (two-step method)

[0026] a) contacting a sample containing bacterial cells or cellcomponents with bacteriophages and/or bacteriophage proteins, preferablywith an incubation time of about 3-5 minutes,

[0027] b) subsequent incubation of said sample, containing the bacterialcells or cell components and the bacteriophages and/or bacteriophageproteins with a solid support, preferably for about 3-30 minutes,

[0028] c) separation of the solid support with the bacterial cells orcell components bound via the bacteriophages and/or bacteriophageproteins to said solid support from the sample.

[0029] Bacterial cells or cell components may be enriched selectivelywith methods according to the present invention, for example from mixedcultures of different species or from a culture of a single species.Enriched cell components may be, for example, endotoxines, proteins,nucleic acids, or saccharides. The choice of appropriate bacteriophagesand/or bacteriophage proteins allows the selectivity of the method.According to the method of the present invention, bacteriophages and/orbacteriophage proteins are most suited for a selective enrichment ofbacteria or cell components, because phage-bacteria-systems have beenevolved in nature for a long time so that the phages identify their hostbacteria in a highly specific manner and with high binding affinity.Preferably bacteriophage proteins are used for the method of the presentinvention which are specific for the bacteria desired to be detected.Bacteriophages as well as bacteriophage proteins developed under adverseenvironmental conditions so that they are stable over influences, liketemperature and pH-variations (Burda et al., Biological Chemistry 2000,381, 255-258) et al., and thus may be used in the different purificationbuffers.

[0030] Which bacteriophages and/or bacteriophage proteins will be useddepends on the fact which bacteria species are to be purified. For afollowing plasmid purification, those bacteriophages and/orbacteriophage proteins will be preferred which may bind the E. colibacteria selectively, because they represent the commonly used bacteriafor a plasmid preparation at present. A large number of knownbacteriophages is available already for most of the bacteria describedso far and may be utilised for a selective bacteria enrichment. Thefollowing table shows an overview of bacteria and their specificbacteriophages without being exhaustive. The phages and their respectivehost bacteria are commercially available from the following straincollections: ATCC (USA), DSMZ (Germany), UKNCC (Great Britain), NCCB(Netherlands), and MAFF (Japan). Moreover, bacteriophages directedagainst respective bacteria may be isolated for example fromenvironmental samples according to standard methods, if required(Seeley, N. D. & Primrose, S. B., 1982, J. Appl. Bacteriol. 53, 1-17).Bacteria: Phage Acholeplasma: 0c1r, 10tur, L2, L51, M1, MVG51, MV-L1,O1, SpV1, V1, V1, V2, V4, V5 Actinomycetes: 108/016, 119, 29, 37, 43,51, 59.1, A1-Dat, Aeh2, Bir, M1, MSP8, ø115-A, ø150A, ø31C, P-a-1, PhiC,R1, R2, SK1, SV2, VP5 Actinoplanes/Micro- Ap3, Ap4, Mm1, Mm3, Mm4, Mm5,phiUW monospora: 51 Aeromonas: 43, 44RR2.8t, 65, Aeh1 Aeromonashydrophila: PM1 Agrobacterium: PIIBNV6, PS8, psi, PT11 Alcaligenes:8764, A5/A6, A6 Alteromonas: PM2 Amycolatopsis: W11, W2, W4, W7Bacillus: 1A, alpha, AP50, BLE, F, G, GA-1, II, IPy-1, mor1, MP13, MP15,ø105, ø29 (phi 29), øNS11, PBP1, PBS1, SP10, SP15, SP3, SP8, SPP1, SPβ,SPy-2, SST, type Bacillus subtilis 168, W23, SP50, W23, SP01Bdellovibrio: MAC-1, MAC-2, MAC-4, MAC-5, MAC-7 Brucella: TbCaulobacter: øCb12r, øCb2, øCb23r, øCb4, øCb5, øCb8r, øCb9, øCP18, øCP2,øCr14, øCr28 Cellulomonas: O11, O13, O2, O3, O5, O6, O8 Chlamydia: 1Chlamydia psittaci: .phi.CPG1 Clostridium: Ceβ, F1, HM2, HM3, HM7Coryneforme 7/26, A, A19, AN25S-1, Arp, AS-1, BL3, CONX, MT, N1, øA8010,S-6(L), β, Cyanobacteria: A-4(L), AC-1, LPP-1, S-2L, S-4L, SM-1 E. coli,(O157): P1, T1, Tula, Tulb, Tull E. coli: 1ø3, 1ø7, 1ø9, 2D/13, Ae2,alpha10, alpha3, BE/1, BF23, dA, delta1, delta6, dø3, dø4, dø5, Ec9,eta8, f1, fd, G13, G14, G4, G6, HK022, HK97, HR, lambda, M13, M13mp18,M20, MM, MS2, Mu, O1, ø80, øA, øR, øX174, PA-2, P1, P1D, P2, P22, Qβ,R17, S13, St-1, T1, T2, T3, T4, T5, T6, T7, WA/1, WF/1, WW/1, zeta3,ZG/2, ZJ/2 E. coli R1: C21 E. coli O8: omega 8 E. coli (K12): U3Enterobacter: chi, FC3-9, μ2, 01, 11F, 121, 1412, 3, 3T+, 50, 5845, 66F,7480b, 8893, 9, 9266, a1, alpha15, b4, B6, B7, Beccles, BZ13, C-1, C16,C2, C-2, DdVl, Esc-7-11, f2, fcan, FI, Folac, fr, GA, H, H-19J, I2-2,Ialpha, ID2, If1, If2, Ike, JP34, JP501, K19, KU1, M, M11, M12, MS2,NL95, ø92, øl, Øii, Omega8, pilHalpha, PR64FS, PRD1, PST, PTB, R, R17,R23, R34, sd, SF, SMB, SMP2, SP, β, ST, tau, tf-1, TH1, TW18, TW28,Vill, VK, W31, X, Y, ZG/1, ZIK/1, ZJ/1, ZL/3, ZS/3 Klebsiellapneumoniae: AP3, C3: Lactobacillus: 1b6, 223, fri, hv, hw222a, øFSW,PL-1, y5 Lactococcus lactis: 1, 643, c2, kh, ml3, P008, P127, 1358,1483, 936, 949, BK5-T, c2, KSY1, P001, P008, P107, P335, PO34, PO87Leuconostoc: pro2 Listeria: 4211, Methanothermobacter: psi M2 (ψM2)Micrococcus: N1, N5 Mollicutes: Br1, C3, L3 Mycobacterium: I3,lacticola, Leo, ø17, R1-Myb Nocardia/Rhodococcus/ N13, N18, N24, N26,N36, N4, N5 Gordona: Nocardioides: X1, X10, X24, X3, X5, X6, D3, D4,Pasteurella: 22, 32, AU, C-2 Promicromonospora: P1, P2, P3, P4Pseudomonas aeruginosa: Phi CT, phi CTX, PB-1 Pseudomonas: 12S, 7s, D3,F116, gh-1, gh-1, Kf1, M6, ø1, øKZ, øW-14, Pf1, Pseudonocardia: W3Rhizobium: 2, 16-2-12, 2, 317, 5, 7-7-7, CM1, CT4, m, NM1, NT2, ø2037/1,ø2042, øgal-1-R, WT1 Saccharomonospora: Mp1, MP2 Saccharothrix: W1Salmonella: epsilon15, Felix 01, 16-19, 7-11, H-19J, Jersey, N4, SasL1,Vil, ZG/3A, San21 Salmonella typhimurium: A3, A4, P22 Spiroplasma: 4,C1/TS2 Sporichthya: Sp1 Staphylococcus: 107, 187, 2848A, 3A, 44AHJD, 6,77, B11-M15, Twort Streptococcus: 182, 2BV, A25, A25-24, A25-omega8,A25-PE1, A25-VD13, CP-1, Cvir, H39 Streptomyces: P23, P26, phi A.streptomycini III, phi8238, phiC31, S1, S2, S3, S4, S6, S7, SH10Terrabacter: Tb1, Tb2 Tsukamurella: Ts1 Vibrio: 06N-22P, 06N-58P,06N-58P, 4996, alpha3alpha, I, II, III, IV, kappa, nt-1, OXN-100P,OXN-52P, v6, Vf12, Vf33, VP1, VP11, VP3, VP5, X29 Xanthomonas: Cf, Cf1t,RR66, Yersinia: 8/C239, phiYeO3-12, YerA41

[0031] If single phage proteins instead of bacteriophages are used,there is an advantage because in this case the properties of a singleprotein instead of a complex of proteins and nucleic acids may be used.Phage proteins are very stable (Burda et al., Biological Chemistry 2000.381, 255-258); the stability of a single protein is much easier tocontrol than the stability of a protein complex. In comparison tocomplete phages, it is important that they are easier to modify(genetically, but also chemically), for example the introduction oftags. Moreover, the use of phage proteins is an advantage in specificconnecting methods, i.e. the isolation of nucleic acids (plasmid DNA,RNA, genomic DNA), because compared to the use of complete phages nonucleic acid contamination is possible.

[0032] Preferred are phage tail proteins from the family of myoviridae,of podoviridae, and siphoviridae, particularly short phage tailproteins, particularly the short phage tail proteins of theeven-numbered T-phages, for example T4, T2, or K3, particularly thebacteriophage tail proteins p12 from T4, p12 from T2 (GenBank AccessionNumber X56555), p12 from K3 (cf. Burda et al., 2000, Biol. Chem., Vol.381, pp.255-258) or the bacteriophage tail proteins from the phagesFelix 01, P1, or PB1. As an example, the short bacteriophage tailproteins of the phages T4 (p12) and from P1 bind to coliformes, theshort phage tail protein from Felix 01 binds to salmonellas, and theshort phage tail protein from PB1 binds to pseudomonads.

[0033] Phage tail proteins like p12 or P22 tailspike protein display ahigh stability over proteases, detergents, chaotropic agents, forexample urea or guanidinium hydrochloride, or higher temperatures(Goldenberg, D. und King, J.; Temperature-sensitive mutants blocked inthe folding or subunit assembly of the bacteriophage P22 tail spikeprotein. II. Active mutant proteins matured at 30° C., 1981, J. Mol.Biol. 145, 633-651. Miller, S., Schuler, B. und Seckler, R.; Phage P22tailspike: Removal of headbinding domain unmasks effects of foldingmutations on native-state thermal stability, 1998, Prot. Sci. 7,2223-2232.; Miller, S., Schuler, B. und Seckler, R.; A reversiblyunfolding fragment of P22 tailspike protein with native structure: Theisolated β-helix domain, 1998, Biochemistry 37, 9160-9168.; Burda etal., 2000, Biol. Chem., Vol. 381, pp. 255-258). The removal of the phagehead and phage base plate binding region, respectively, of theseproteins may reduce a potentially existing aggregation sensitivity.Interestingly, the single domains and subunits, respectively, of theseproteins are significantly less stable than the intact or onlymarginally reduced trimers (Miller et al., Prot. Sci. 1998; 7:2223-2232. Phage P22 tailspike protein: Removal of head-binding domainunmasks effects of folding mutations on native-state thermal stability.;Miller-S, et al., Biochemistry 1998; 37: 9160-9168. A reversiblyunfolding fragment of P22 tailspike protein with native structure: Theisolated β-helix domain). Furthermore the single domains and subunits,respectively, are presumably hardly stable and functionally expressable:phage tail proteins and virus receptor proteins are often available asintensely drilled trimers, which has been shown in crystallographicexperiments wherein the C-terminus may fold back, which is a mechanismpossibly providing an additional protection against proteases (MitrakiA, Miller S, van Raaij M J. Review: conformation and folding of novelBeta-structural elements in viral fibre proteins: the triple Beta-spiraland triple Beta-helix. J Struct Biol. 2002 137(1-2):236-247), Moreover,these proteins exist in the native condition as homotrimers. The trimerscontribute with three binding sites to a stronger binding of bacteria byan increase of the avidity.

[0034] With the even-numbered T-phages (T4, T2, K3) as an example, thebinding mechanism of the bacteriophage proteins to the single bacteriashould be clarified. In this genus, there are two components on the hostside which are recognised by the phages: firstly a surface proteinspecific for individual phages, secondly the lipopoysaccharide (LPS)which is possessed by all gram-negative bacteria in a modified form ontheir outside and is orientated to the envrionment. The long tail fibresof the even-numbered T-phages play a role in the specific recognition ofthe host bacteria, whereas the LPS serves as a receptor for the shorttail fibres. It is known from the phage T4 from E. coli that thespecific interaction with the host bacterium mediated by the long tailfibres will become irreversible as soon as the short tail fibres havebeen bound to the bacteria surface. The short tail fibre is notresponsible for the correct specificity within the host bacteria genusand therefore may be replaced between the different even-numberedT-phages.

[0035] Bacteriophage tail proteins may easily be recombinantly producedin large numbers and may be purified using appropriate tags or simplechromatographic standard separation methods. Phages as well as hoststrains are largely commercially available via strain collections or maybe isolated by simple means. In the method of the present invention,however, not only the naturally occurring bacteriophage tail proteinsmay be used, but also their variants. The variants as used in thepresent invention means that the bacteriophage tail proteins exhibit analtered amino acid sequence. Said variants may be obtained by screeningof the naturally occurring variants or by random mutagenesis or targetedmutagenesis, but also by chemical modification. The bacteriophage tailproteins used in the method of the present invention may be adapted by atargeted or random mutagenesis in their host specificity and theirbinding behaviours, respectively, to the support structures. By means ofthe mutagenesis, mutations are introduced which may be amino acidadditions, deletions, substitutions, or chemical modifications. Thesemutations produce an alteration of the amino acid sequence in thebinding region of the phages or phage proteins, with the intention toadapt the specificity and binding affinity to the experimentalrequirements, for example to enhance the binding of the bacteria to theisolated phage proteins or to make their binding irreversible, toenhance the washing options. Moreover, a genetic or biochemicalmodification of the phage proteins may be performed with the intentionoptionally to switch off the present enzymatic activity to improve thebinding or make the binding irreversible.

[0036] For binding purposes of the bacteria and/or cell components to bepurified to the bacteriophages and/or bacteriophage tail proteins in thetwo-step method, the sample, for example an overnight culture, iscontacted with the bacteriophages and/or bacteriophage tail proteins andis preferably incubated. The incubation occurs at a temperature in therange of 4° C. to 90° C., preferably at a temperature in the range of 4°C. to 45° C., more preferred at a temperature in the range of 15° C. to37° C., furthermore preferred at a temperature in the range of 20° C. to37° C., in particular at RT, for up to 6 hours, preferably up to 4hours, more preferred 2 hours, in particular 1 hour, in particularpreferred 1-20 minutes, exceptionally preferred 3-5 minutes. Forexample, the incubation can occur for 2 to 120 minutes at 4° C. to 37°C., preferably for 20 to 30 minutes at 25° C. to 37° C., preferably morepreferred for 3-5 minutes at 37° C.

[0037] The sample is contacted with solid supports subsequently andincubated. Solid supports may be, for instance, magnetic particles(paramagnetic or ferromagnetic), glass particles, agarose particles,luminex particles, reactions tubes, or microtiter plates.

[0038] In case of using magnetic particles, they were subsequently addedto the sample. The magnetic particles bind thebacteriophage/bacteriophage protein-bacteria/cell component complex,which is then easily separated from the sample by using magnetic means,and which may then be purified. The magnetic means may be positioned atthe outside of the container and either may be switched on for theenrichment so that the magnetic particles are collected at the containerwall, or may slide along the outside wall of the container so that themagnetic particles are collected e.g. at the bottom of the container.The enrichment with a permanent magnet is preferred. The magnetic meansmay also immerse into the container and the sample so that the magneticparticles deposit at the magnetic means (the magnetic means may becovered by a pipette tip or a comparable disposable). In comparison tocentrifugation or filtration techniques, the bacteria are subject toonly minimal shear rates and therefore may be enriched with high yieldin an active/living manner, if required. The easy handling facilitateseasy and fast buffer/solution changes and may both easily be performedon a large scale, and well automated.

[0039] The magnetic particles exhibit a diameter allowing the binding ofa sufficient amount of cells or cell components per particle. Preferablythe magnetic particles exhibit a diameter in the range of about 0.5 toabout 4 μm, in particular in the range of about 0.5 to about 2 μm, morepreferred in the range of about 0.8 to about 1.8 μm, most preferredabout 1 μm.

[0040] The binding of the bacteriophage/bacteriophageprotein-bacteria/cell component complexes to the solid supports, forexample magnetic particles, preferably occurs via appropriate couplinggroups, in particular polypeptides and/or low molecular substances.These polypeptides may also be antibodies, lectins, receptors oranticalins specific for the bacteriophages and/or bacteriophageproteins. Furthermore, the bacteriophages and/or bacteriophage proteinsmay be coupled to low molecular substances, e.g. biotin, to bind topolypeptides, e.g. streptavidin, via these low molecular substanceswherein the polypeptides may be immobilised to the support. Instead ofbiotin, the so-called strep-tag (Skerra, A. & Schmidt, T. G. M.Biomolecular Engineering 16 (1999), 79-86) may be used, which is a shortamino acid sequence and binds to Streptavidin. Furthermore, the his-tagmay be used, which may bind to a support material via bivalent ions(zinc or nickel) or an antibody which is specific for the his-tag(Qiagen GmbH Hilden, Germany). The strep-tag as well as the his-tag arepreferably bound by means of DNA recombination technology to therecombinantly produced bacteriophage proteins. This coupling may occurin a directed manner, e.g. to the N- or C-terminus. Since particularlyin the two-step method a high binding constant is essential for aneffective enrichment, the coupling combination of biotin/streptavidinwith a kD of ˜10⁻¹⁵M (Gonzales et al. J. Biol. Chem., 1997, 272 (17),pp. 11288-11294) is preferred in particular. It was shown that thisnon-covalent binding combination works better than the availableantibodies, anticalins, receptors and lectins.

[0041] For a binding of the complex the magnetic particles are contactedwith the bacteriophage/bacteriophage protein-bacteria/cell componentcomplex and are preferably incubated. The incubation occurs at atemperature in the range of 4° C. to 90° C., particularly in the rangeof 4° C. to 45° C., more preferred at a temperature in the range of 15°C. to 37° C., particularly preferred at a temperature in the range of20° C. to 37° C., in particular at RT, for up to 6 hours, preferably upto 4 hours, more preferred 2 hours, in particular 1 hour, in particularpreferred 1-20 minutes, exceptionally preferred 3-5 minutes. Forexample, the incubation can occur for 2 to 120 minutes at 4° C. to 37°C., preferably for 20 to 30 minutes at 25° C. to 37° C., preferably morepreferred for 3-5 minutes at 37° C.

[0042] The method of the present invention is performed with other solidsupports which may be added to the sample in the analogous manner. Thesingle incubation conditions and separation steps have to be adapted forthe different solid supports accordingly. This may easily be performedin test series and does not require any further explanation for theskilled artisan.

[0043] Alternatively, the two-step method may be performed inaccordingly coated solid supports which may not be added to the sample,but wherein the sample is added onto or into the solid support, e.g. amicrotiter plate or a reaction tube. For this purpose, the sample isadded after step a), e.g. into the respective wells of the microtiterplate, and is incubated there, particularly for about 20-30 minutes withthe other conditions remaining as described above. The wells of themicrotiter plate or the inner walls of a reaction tube may exhibit thesame coatings as described above for the magnetic particles.

[0044] The enrichment and purification, respectively, of the bacterialcells and/or cell components may also be performed in a methodcomprising the following steps (one-step method):

[0045] a) contacting a sample containing bacterial cells and/or cellcomponents with a magnetic support on the surface of whichbacteriophages and/or bacteriophage proteins are applied, preferablywith an incubation of about 3-60 minutes,

[0046] b) separating the magnetic support with the bacterial cellsand/or cell components bound to it from the sample.

[0047] The methods according to the present invention (one-step andtwo-step method) may be used, for example, as an alternative forcentrifugation and thus for the first time allows the automatedpurification of bacterial cells. This for the first time enables theautomation of, e.g., the genome analysis, i.e. from the inoculation ofthe bacterial cultures to the determination of the sequence.Furthermore, the method of the present invention may be used, forexample, to isolate cell components, particularly oflipopolysaccharides, endotoxines or exopolysaccharides.

[0048] The following embodiments of the coupling or immobilisation ofbacteriophages and/or bacteriophage proteins to the magnetic particles(one-step method) apply accordingly to the coupling or immobilisation ofbacteriophages and/or bacteriophage proteins and polypeptides to thesolid supports (two-step method). The coating of the solid supports withthe previously described polypeptides or the bacteriophages and/orbacteriophage proteins may occur in a different manner.

[0049] The bacteriophages and/or bacteriophage proteins may be fixed tothe solid supports via covalent coupling. This allows a very tightbinding to the support and thus the application of severe washingconditions for the washing of the cells which is possibly required for afurther processing of the enriched cells. The coupling of thebacteriophages and/or bacteriophage proteins via adsorption is a verysimple and cost-effective method. One-step as well as two-step methodsare possible by means of coupling the bacteriophages and/orbacteriophage proteins via biotin/streptavidin or comparableligand/receptor systems. The streptavidin used in this approach may befixed via adsorption, as well as via chemical coupling. A functionalimmobilisation is important in the coating method, that means thatdespite their binding to the solid supports, the bacteriophages and/orbacteriophage proteins exhibit structures which are accessible tobacteria.

[0050] The bacteriophages and/or bacteriophage proteins may be coupledvia covalent coupling to support materials which have already beenactivated by the manufacturers, for instance to magnetic particles byMerck, Estapor, etc. via standard conditions, for example —NH₂ viacyanuryl chloride (Russina Chemical Rev., 1964, 33: 92-103), or —COO—via EDC (1-Ethyl-3′[3′Dimethylaminopropyl]carbodiimid) (Anal. Biochem.1990, 185: 131-135). Moreover, the solid supports may be activateddirectedly using appropriate methods. Furthermore, the coupling mayoccur via maleimide or iodoacetyl spacer to, for instance, a N-terminalintroduced cystein.

[0051] The immobilisation of the bacteriophages and/or bacteriophageproteins to the support material via adsorption may be performed byincubation of a bacteriophage and/or bacteriophage protein solution inaqueous buffer, for instance 100 mM Tris pH 7.3, or 100 mM sodiumphosphate pH 7.5, PBS (10 mM sodium phosphate pH 7.4, 150 mM sodiumchloride) for several hours or overnight at 4° C. to 45° C., preferablyat 15° C. to 37° C., more preferred at 20° C. to 37° C., in particularpreferred at 37° C. or RT, in particular preferred at 30° C. to 65° C.for 2-4 hours. The coating solution is discarded after the adsorptionand the support structure is stored in aqueous, optionally in bufferedsolution.

[0052] A further aspect of the present invention is a solid support, inparticular a magnetic particle or a microtiter plate, either coated withbacteriophages and/or bacteriophage proteins, or coated withpolypeptides directed against bacteriophages and/or bacteriophageproteins. These polypeptides may be antibodies, lectins, receptors oranticalins specific for the bacteriophages and/or bacteriophageproteins. The solid supports may be coated furthermore withstreptavidin.

[0053] A further aspect of the present invention are bacteriophageproteins coupled with so-called tags, for example the strep- or thehis-tag, particularly to the 3′- or 5′ terminus, more preferred to the5′ terminus. The coupling or linking of the tags with the bacteriophageproteins via DNA recombination technology is preferred. The productionof the nucleic acid, comprising the sequence of the bacteriophageprotein and the tag, and the production of the expression product arestate of the art and there is no need to explain the production indetail at this point. A further aspect of the present invention is thenucleic acid sequence coding the bacteriophage protein together with thestrep- or the his-tag. A p12 protein from the bacteriophage T4 is aparticularly preferred bacteriophage protein modified with the strep- orthe his-tag, however, all other bacteriophage proteins of the listedbacteriophages from the above table are also preferred.

[0054] A further aspect of the present invention are bacteriophageproteins with a tag exhibiting a surface-exposed cysteine for thespecific, directed biotinylation, e.g. the tags according to SEQ ID NOs:5, 6 or 7. One example for a p12 with a tag is the amino acid sequencedepicted in SEQ ID NO: 8. Preferred is a p12 with a tag, in particularwith a tag with a surface-exposed cysteine, in particular a p12 with atag according to SEQ ID NOs: 6 and 7. In addition, said directedbiotinylation may be mediated by an appropriate spacer or linker.Furthermore, the present invention relates to the amino acid sequencesaccording to SEQ ID NOs: 5, 6 and 7. Furthermore, the present inventionrelates to nucleic acids coding for the amino acid sequences accordingto SEQ ID NOs: 5, 6 and 7.

[0055] A further aspect of the present invention relates to a kit forthe enrichment of bacterial cells and/or cell components, comprising thesolid supports according to the present invention, for example themagnetic particles, glass particles, agarose particles, reaction tubesor microtiter plates as well as the solutions including the testreagents necessary for the enrichment of the bacteria and/or cellcomponents.

[0056] The kit for the enrichment with magnetic particles includes inparticular a stabilised solution of a p12-variant with a cysteineresidue for the directed biotinylation introduced at the N-terminus, forexample NS-T4p12 (or T4p12bio) 1 mg/ml in 100 mM Tris HCl pH8, 150 mMNaCl, 1 mM EDTA, 0.05% Tween 20, supplement with a protease inhibitormixture (Sigma) as a solution (preferred storage at 4° C.) or as alyophilisate. Furthermore, the kit includes a particle solutionconsisting of streptavidin- or streptactin-coated magnetic particles ina stabilising solution (PBST with sodium azide 0.005%).

[0057] The kit for an enrichment with microtiter plates includes inparticular a stabilised solution of a p12-variant with a cysteineresidue for the directed biotinylation at the N-terminus, for exampleNS-T4p12 (or T4p12bio) 1 mg/ml in 100 mM Tris HCl pH8, 150 mM NaCl, 1 mMEDTA, 0.05% Tween 20, supplement with a protease inhibitor mixture(Sigma) as a solution (preferred storage at 4° C.) or as a lyophilisate.The kit furthermore includes a streptavidin- or streptactin-coatedmicrotiter plate.

[0058] The following examples illustrate the invention and are not to beunderstood as limiting. If not indicated otherwise, molecular biologicalstandard methods have been used, as for example described by Sambrook etal., 1989, Molecular cloning: A Laboratory Manual 2. Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0059] 1. The Purification of Wild Type T4p12 was Carried Out Accordingto the Method Described in Burda, M. R. & Miller, S. Eur. J. Biochem.1999, 265 (2), 771-778.

[0060] 2. Construction of p12 with a N-Terminal Strep-Tag (N-Strep-p12):

[0061] The nucleotide sequence of the strep-tag (U.S. Pat. No.5,506,121) was introduced to the 5′ terminus of the T4p12 gene via PCR.For the 5′ terminus of the p12 gene a primer was constructed (5′-GAA GGAACT AGT CAT ATG GCTAGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT MT MTACA TAT CM CAC GTT-3′), (SEQ ID NO:1) including the nucleotide sequenceof the strep-tag at its 5′ terminus (printed in italics in the sequence)and having a restriction recognition sequence (NdeI, underlined in thesequence) in such a manner that the gene may be inserted in the correctreading frame into the expression plasmid. A primer was constructed wasconstructed for the 3′ terminus of the p12 gene introducing a BamH1restriction recognition sequence (printed in italics in the sequence)behind the p12 gene (5′-ACG CGC AAA GCT TGT CGA CGG ATC CTA TCA TTC TTTTAC CTT MT TAT GTA GTT-3′), (SEQ ID NO:2). The PCR was performed with 40cycles (1 min 95° C., 1 min 45° C., and 1 min 72° C.). The PCRpreparation was cleaved with the restriction endonucleases NdeI andBamH1 and the desired fragment was inserted in the NdeI and BamH1 siteof the expression plasmid pET21a (Novagen, Merck Eurolab, Darmstadt, DE)after size directed separation via an agarose gel and elution from thegel. The sequence of the N-strep-p12 gene was verified by DNAsequencing. The further steps to the plasmid pNS-T4p12p57 were performedas described by Burda, M. R. & Miller, S. (Eur. J. Biochem. 1999, 265(2), 771-778). The plasmid pNS-T4p12p57 was then transformed into theexpression strain BL21 (DE3) (Novagen, Merck Eurolab, Darmstadt, DE).

[0062] 3. Introduction of a N-Terminal Cysteine Residue in N-Strep-p12(N-Strep-S3C-p12 and N-Strep-S14C-p12):

[0063] The introduction of a N-terminal cysteine residue (bold type) wasperformed as described under 2. above with two new primers for the 5′terminus being constructed. The primer 5′-GM GGA ACT AGT CAT ATG GCT TGTTGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT MT MT ACA TAT CM CAC GTT-3′(SEQ ID NO:3) was used for the N-strep-S3C-p12, and the primer 5′-GM GGAACT AGT CAT ATG GCTAGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC TGT MTAAT ACA TAT CM CAC GTT-3′ (SEQ ID NO:4) was used for the N-strepS14C-p12.

[0064] 4. Purification of N-Strep-p12 Protein:

[0065] The E. coli strain BL21 (DE3) with the plasmid pNS-T4p12p57 wascultured in 21 shaking cultures (LB medium with ampicillin 100 μg/ml) upto an OD600 of 0.5-0.7 at 37° C. and the expression of the N-strep-p12protein was induced by adding 1 mM IPTG(Isopropyl-β-thiogalactopyranoside). The cells were harvested afterincubation at 37° C. for 4 hours. Harvested cells from a 10 l culturewere resuspended in 50 ml sodium phosphate, 20 mM pH 7.2, 2 mM MgSO₄,0.1 M NaCl, broken up by a triple French press treatment (20,000 psi),and were subsequently centrifuged for 30 minutes at 15,000 rpm (SS34).After washing it twice in the same buffer, the N-strep-p12 protein wasextracted from the pellet by stirring for 30 minutes in 40 mM Tris HClpH 8.0, 10 mM EDTA. The preparation was centrifuged for 30 minutes at15,000 rpm (SS34) and the supernatant with the separated NS-p12 wasstored at 4° C. The extraction was repeated twice and the combinedsupernatants were applied to a streptactin-affinity column (15 ml)equilibrated with buffer “W” (100 mM Tris HCl pH 8, 1 mM EDTA, 150 mMNaCl) (IBA GmbH, Göttingen, D E). After washing with 5 volumes of thecolumn with buffer “W”, the column was eluted with 3 volumes of buffer“W” with 2.5 mM desthiobiotin in buffer “W”. After repeated dialysiswith “W” and concentration, the concentration and purity ofN-strep-T4p12 was determined via SDS-PAGE and UV Spectroscopy (Burda etal. 1999). In this way, about 100 mg N-strep-T4p12 were purified from a10 l culture. Name Sequence of the tag Nstrep-p12 MASWSHPQFEKGAS SEQ IDNO: 5 Nstrep-p12-S3C MACWSHPQFEKGAS SEQ ID NO: 6 Nstrep-p12-S14CMASWSHPQFEKGAC SEQ ID NO: 7

[0066] 5. Alternative Purification of p12:

[0067] The cell pellet (from a 10 l culture, BL21 (DE3), transformedwith the plasmid pNS-T4p12p57 or pT4p12p57) was resuspended in buffer 1(10 mM sodium phosphate pH 9, 500 mM NaCl, 4 mM MgCl₂) and broken up viaFrench press (as described under 3.). Subsequently the material wascentrifuged at 20,000 rpm (SS34) for 45 minutes and the pellet wasresuspended (i.e. washed) in about 25 ml of buffer 1. This washing stepwas repeated twice and the pellet was extracted with 25 ml of buffer 2:(50 mM NaPi pH 5,100 mM NaCl, 25 mM EDTA). The resuspended pellet wasstirred for the extraction for 60 minutes at RT and afterwardscentrifuged (20,000 rpm (SS34) for 45 minutes). Said extraction wasrepeated twice if required. Afterwards, the supernatants of theextraction were combined and applied directly to an anion exchangecolumn (ResoureceS, Pharmacia). 15 mM sodium hydrogen phosphate, 15 mMsodium formiate, 30 mM sodium acetate, pH 5.0, 50 mM NaCl were used as arunning buffer. The elution occurred via a linear salt gradient of 50 mMNaCl to 60 mM NaCl in the running buffer. The purified p12 wassubsequently dialysed for storage against 50 mM Tris pH 8.5, 150 mMNaCl, 5 mM EDTA or PBS, frozen in aliquots in N₂, and stored at −20° C.

[0068] 6. Biotinylation of p12:

[0069] For the biotinylation of the p12 protein, eitherEZ-Link™Sulfo-NHS-LC-LC-Biotin or EZ-Link™TFP-PEO-Biotin by Pierce, USA,was used or the biotinylation was performed according to the methods ofthe manufacturer. About 30 molecules of biotin per p12-trimer were usedfor the biotinylation. The coupling of biotin was subsequently verifiedwith a HABA-assay (Savage M D, Mattson G, Desai S, Nielander G W,Morgensen S, and Conklin E J, 1992, Avidin-Biotin Chemistry: A Handbook,Pierce, Ill.) and quantified. Finally, an average of 5-10 molecules ofbiotin per p12-trimer were bound.

[0070] 7. For the Biotinylation of the N-Terminal Introduced Cysteine,EZ-Lin™PEO-Maleiimid-Biotin and EZ-Link™PEO-Liodoacetyl-Biotin byPierce, USA, were Used According to the Instructions of themanufacturer. The Reaction was Verified as Described under 5. Above.

[0071] 8. Biotinylation of the Bacteriophage T4:

[0072] The bacteriophage was purified according Bachrach U and FriedmannA (1971) Practical procedures for the purification of bacterial viruses,Appl. Microbiol 22: 706-715. The purified phage was dialysed against PBSand labelled with EZ-Link™Sulfo -NHS-LC-LC-Biotin orEZ-Link™TFP-PEO-Biotin by Pierce, USA, according to the instructions ofthe manufacturer in a ratio of 100-100,000 biotin molecules per phage.

[0073] 9. p12-Dependent Harvest of E. Coli According to the One-StepMethod and the Two-Step Method (FIG. 1):

[0074] In the binding step according to the one-step method (VC) theN-strep-p12 protein was incubated for 1 hour with the magneticstreptavidin beads (10 μg protein/50 μl 1% beads) and the beads weresubsequently washed three times with PBST. For the cell binding step,200 μl of an E. coli overnight culture diluted 1 to 10 in LB (about1×10⁸ cells/ml) per well in a microtiter plate were mixed with 10 μl ofthe N-strep-p12 coated beads and incubated for different periods of timeat RT (FIG. 1). The beads were concentrated subsequently with the boundcells via a magnetic separator (Bilatec A G, Mannheim, D E) for 3-5minutes at the walls of the wells. The beads were washed three timeswith PBST and the β-galactosidase activity (Apte S C et al., 1995, Waterres. 29, 1803-06) of the cells attached to the beads were determinedsubsequently. For the binding step according to the two-step method, a 1to 10 dilution of an E. coli overnight culture (about 1×10⁸ cells/ml)were incubated for 1 hour (in further approaches for 1 minute, 3minutes, 10 minutes, 30 minutes) with the N-strep-p12 protein (10 μgprotein/ml cell suspension) at RT. Subsequently, 200 μl of theprotein-cell-mixture were added to 10 μl of 1% magnetic streptavidinbeads and incubated at RT for the times given in FIG. 1. Thedetermination of the bound cells was performed according to the one-stepmethod.

[0075] 10. T4-Dependent Binding of E. Coli According to the One-StepMethod (VC) and the Two-Step Method (VIK) (FIG. 2)

[0076] In the binding step according to the one-step method (VC), thebiotinylated phage T4 (100 biotin molecules/phage) were bound for 1 hourto 1% streptavidin beads (10¹⁰ PFU/ml 1% beads) and the beads werewashed three times with PBST subsequently. After 2 hours of the cellbinding step (25 μl phage beads/ml cell suspension), the beads werewashed and the bound E. coli were determined via the β-galactosidaseactivity (the data are given in relative units). In the binding stepaccording to the two-step method (VIK), the cells were incubated withthe biotinylated phages for 1 hour (2.5×10⁸ PFU/ml cell suspension) andthe mixture was incubated subsequently for 2 hours with the streptavidinbeads (25 μl 1% beads/ml cell suspension). The further steps wereperformed according to the one-step method. During the continuousincubation of phage and bacteria, the antibiotics Rifampicin (25 μg/ml),Chloramphenicol (25 μg/ml) and Tetracycline (2 μg/ml) were added to themedium.

[0077] 11. Harvest of E. Coli Cells According to the Two-Step MethodFrom Different Growth Media (FIG. 3):

[0078] The E. coli strains LE392 and JM83 were grown overnight in therespective media. N-strep-p12 (10 μg/ml cell suspension) were added to200 μl of cell suspension. 10 μl 1% streptavidin beads were added after5 minutes of incubation at RT, mixed by pulling it three times with apipette, and were incubated a further 5 minutes at RT. Subsequently, thebacteria beads complexes were removed by means of the above describedmagnetic separator for 3-5 minutes and the cells remaining in thesupernatant were determined via the scattering of the supernatant at 600nm.

[0079] 12. Plasmid Isolation of E. coli After Cell Harvest via theTwo-Step Method (FIG. 4).

[0080] 300 μl each of a E. coli overnight culture containing the plasmidpUC19 were harvested according to the two-step method as described underexample 9. After removal of the cells by means of the above describedmagnetic separator, the plasmid was isolated from the cells in a firstmethod via the solid phase extraction methods (QIAprep, Qiagen, Hilden,D E) and also via a method using magnetic beads (Bilatec, Mannheim, D E)according to the instructions of the manufacturers.

[0081] 13. Enrichment of E. Coli Cells from 10 ml Culture Volume VIAN-Strep-p12 and via the Biotinylated Bacteriophage T4-Bio According tothe Two-Step Method (FIG. 5):

[0082] For the enrichment with N-strep-p12, 10 ml of E. coli cultureswith 10⁹, 10⁸ and 10⁷ cells per ml were mixed with 30 μg and 6 μgprotein each, respectively, and scrolled for 1 hour at 37° C. For theenrichment with T4-bio, 10 ml cultures with 10⁹, 10⁸ and 10⁷ cells perml were each added to 10¹⁰ T4-bio phages and 10 μg Rifampicin/ml and 2μg Tetracycline/ml and scrolled for 1 hour at 37° C. Subsequently, 100μl 1% magnetic streptavidin beads were added to the preparations andscrolled for 1 more hour. By means of a magnet (ABgene, Hamburg, DE),the cells bound to the magnetic beads were separated, washed three timeswith PBST, and determined via their β-galactosidase activity.

[0083] 14. Living Harvest of E. Coli via the Two-Step Method withBiotinylated p12 (FIG. 6):

[0084]E. coli cells (200 μl of a culture) were harvested according tothe two-step method as described in example 9 (10 μg biotinylated p12/mlcell culture and 10 μl 1% streptavidin beads/ml cell culture) and thebeads washed twice with PBST. Subsequently, the β-galactosidase activityand the growth of the cells after 2 hours was determined via thescattering at 600 nm in a photometer.

[0085] 15. Selectivity of the N-Strep-p12-Dependent Harvest (FIGS. 7 and8):

[0086] 200 μl each of an overnight culture of different E. coli strains(FIG. 7) as well as of different bacteria strains (FIG. 8) wereharvested according to the two-step method (as described in example 9).After concentration of the beads in a magnetic separator, the separatedcell amount was determined via the scattering of the supernatant at 600nm.

[0087] 16. Selective Binding of E. coli to Magnetic Streptavidin Beadsvia T4-Bio and Assay of E. Coli via FITC-Labelled p12 (FIG. 9):

[0088] The p12 protein was labelled with FITC (Molecular Probes, Leiden,N L) according to the instructions of the manufacturer and dialysedagainst PBS. FITC-labelled p12 (5 μg/ml) was added to a mixed culture(about 10⁸⁻⁹ cells/ml) from E. coli and Serratia marcescens. After 5minutes of dark incubation at RT, 10⁹ T4-bio phages and Rifampicin (10μg/ml), Chloramphenicol (25 μg/ml) and Tetracycline (2 μg/ml) wereadded. As a control, non-biotinylated T4 phage was added. After anincubation of 10 minutes, 1% streptavidin beads (10 μl/ml) were addedand the preparations were studied under a microscope (FIG. 9).

[0089] 17. Determination of the Detection Limit for theN-Strep-p12-Dependent Isolation of E. Coli Cells According to theTwo-Step Method (FIG. 10):

[0090] 300 μl each of dilutions of an E. coli overnight culture(10²-10¹⁵ cells/ml) were incubated in microtiter plates with N-strep-p12(10 μg protein/ml) for 1 hour.

[0091] Subsequently magnetic streptavidin beads (100 μl 1% beads/ml)were added, mixed, and the bound cells were pelleted by means of amagnet (Bilatek, Mannheim, D E). The beads were washed three times withPBST. The detection of the E. coli cells occurred via fluorescence andluminescence substrates for the β-galactosidase.

[0092] 18. Chemical Coupling of T4p12 to Magnetic Beads (FIG. 11):

[0093] 150 μl 1% magnetic beads (EM2-100/40, Merck Eurolab, France) werewashed three times with 10 mM sodium phosphate buffer, pH 6, resuspendedin 40 μl of the buffer. After adding 120 μl EDC-solution(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30 mg/ml) andincubation for 5 minutes at RT, 160 μl of T4p12 solution (0.7 mgprotein/ml 10 mM sodium phosphate buffer, pH 6) was pipetted to thesolution, mixed, and the preparation was incubated for 2 hours at RT. Byadding 1 volume of 0.2 M Tris-HCl, pH 7, 0.05% Tween 20, the reactionwas stopped at 4° C. overnight. The beads were washed subsequently 4times with PBST and adjusted to 1% with PBST. The coupling of p12 to thebeads was determined via a p12-specific antiserum. The binding activityof the p12 beads was determined via the binding of E. coli cellsaccording to the one-step method. 3 μl 1% p12 beads were incubated with200 μl of a hundredfold diluted E. coli overnight culture (about 1×10⁷cells/ml) for 5 minutes, washed three times with PBST, and the boundcells were detected via their β-galactosidase activity (FIG. 11).

[0094] 19. Adsorption of p12 to Different Magnetic Polyvinylalcohol(PVA) Beads (FIG. 12):

[0095] 200 μl 2% PVA beads (Chemagen, A G, Baesweiler, D E) wereincubated with different amounts of T4p12 (0-5 μg protein/mg beads) in100 mM Tris HCl, pH 8, 1 mM EDTA, 200 mM NaCl overnight at 37° C. Thebeads were subsequently washed two times with PBST and resuspended inPBS to give 2%. The functional binding of T4p12 to the beads wasdetermined via the binding of E. coli cells. 200 μl of an E. coliovernight culture were mixed with 10 μl of p12 beads and incubated for 5minutes at RT. After removal of the bound cells, the scattering of thesupernatant was measured at 600 nm and set in relation to the scatteringof the E. coli culture before adding the beads.

1 8 1 78 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 gaaggaacta gtcatatggc tagctggagc cacccgcagttcgaaaaagg cgccagtaat 60 aatacatatc aacacgtt 78 2 54 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 2acgcgcaaag cttgtcgacg gatcctatca ttcttttacc ttaattatgt agtt 54 3 78 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer3 gaaggaacta gtcatatggc ttgttggagc cacccgcagt tcgaaaaagg cgccagtaat 60aatacatatc aacacgtt 78 4 78 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 4 gaaggaacta gtcatatggc tagctggagccacccgcagt tcgaaaaagg cgcctgtaat 60 aatacatatc aacacgtt 78 5 19 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide5 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Ser Asn Asn 1 5 1015 Thr Tyr Gln 6 19 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 6 Met Ala Cys Trp Ser His Pro Gln Phe Glu LysGly Ala Ser Asn Asn 1 5 10 15 Thr Tyr Gln 7 19 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 7 Met Ala Ser TrpSer His Pro Gln Phe Glu Lys Gly Ala Cys Asn Asn 1 5 10 15 Thr Tyr Gln 8539 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 8 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Ser AsnAsn 1 5 10 15 Thr Tyr Gln His Val Ser Asn Glu Ser Arg Tyr Val Lys PheAsp Pro 20 25 30 Thr Asp Thr Asn Phe Pro Pro Glu Ile Thr Asp Val Gln AlaAla Ile 35 40 45 Ala Ala Ile Ser Pro Ala Gly Val Asn Gly Val Pro Asp AlaSer Ser 50 55 60 Thr Thr Lys Gly Ile Leu Phe Leu Ala Thr Glu Gln Glu ValIle Asp 65 70 75 80 Gly Thr Asn Asn Thr Lys Ala Val Thr Pro Ala Thr LeuAla Thr Arg 85 90 95 Leu Ser Tyr Pro Asn Ala Thr Glu Ala Val Tyr Gly LeuThr Arg Tyr 100 105 110 Ser Thr Asp Asp Glu Ala Ile Ala Gly Val Asn AsnGlu Ser Ser Ile 115 120 125 Thr Pro Ala Lys Phe Thr Val Ala Leu Asn AsnVal Phe Glu Thr Arg 130 135 140 Val Ser Thr Glu Ser Ser Asn Gly Val IleLys Ile Ser Ser Leu Pro 145 150 155 160 Gln Ala Leu Ala Gly Ala Asp AspThr Thr Ala Met Thr Pro Leu Lys 165 170 175 Thr Gln Gln Leu Ala Val LysLeu Ile Ala Gln Ile Ala Pro Ser Lys 180 185 190 Asn Ala Ala Thr Glu SerGlu Gln Gly Val Ile Gln Leu Ala Thr Val 195 200 205 Ala Gln Ala Arg GlnGly Thr Leu Arg Glu Gly Tyr Ala Ile Ser Pro 210 215 220 Tyr Thr Phe MetAsn Ser Thr Ala Thr Glu Glu Tyr Lys Gly Val Ile 225 230 235 240 Lys LeuGly Thr Gln Ser Glu Val Asn Ser Asn Asn Ala Ser Val Ala 245 250 255 ValThr Gly Ala Thr Leu Asn Gly Arg Gly Ser Thr Thr Ser Met Arg 260 265 270Gly Val Val Lys Leu Thr Thr Thr Ala Gly Ser Gln Ser Gly Gly Asp 275 280285 Ala Ser Ser Ala Leu Ala Trp Asn Ala Asp Val Ile His Gln Arg Gly 290295 300 Gly Gln Thr Ile Asn Gly Thr Leu Arg Ile Asn Asn Thr Leu Thr Ile305 310 315 320 Ala Ser Gly Gly Ala Asn Ile Thr Gly Thr Val Asn Met ThrGly Gly 325 330 335 Tyr Ile Gln Gly Lys Arg Val Val Thr Gln Asn Glu IleAsp Arg Thr 340 345 350 Ile Pro Val Gly Ala Ile Met Met Trp Ala Ala AspSer Leu Pro Ser 355 360 365 Asp Ala Trp Arg Phe Cys His Gly Gly Thr ValSer Ala Ser Asp Cys 370 375 380 Pro Leu Tyr Ala Ser Arg Ile Gly Thr ArgTyr Gly Gly Ser Ser Ser 385 390 395 400 Asn Pro Gly Leu Pro Asp Met ArgGly Leu Phe Val Arg Gly Ser Gly 405 410 415 Arg Gly Ser His Leu Thr AsnPro Asn Val Asn Gly Asn Asp Gln Phe 420 425 430 Gly Lys Pro Arg Leu GlyVal Gly Cys Thr Gly Gly Tyr Val Gly Glu 435 440 445 Val Gln Lys Gln GlnMet Ser Tyr His Lys His Ala Gly Gly Phe Gly 450 455 460 Glu Tyr Asp AspSer Gly Ala Phe Gly Asn Thr Arg Arg Ser Asn Phe 465 470 475 480 Val GlyThr Arg Lys Gly Leu Asp Trp Asp Asn Arg Ser Tyr Phe Thr 485 490 495 AsnAsp Gly Tyr Glu Ile Asp Pro Ala Ser Gln Arg Asn Ser Arg Tyr 500 505 510Thr Leu Asn Arg Pro Glu Leu Ile Gly Asn Glu Thr Arg Pro Trp Asn 515 520525 Ile Ser Leu Asn Tyr Ile Ile Lys Val Lys Glu 530 535

1. A method for the selective purification of bacterial cells or cellcomponents, comprising the following steps: a) contacting a samplecontaining bacterial cells or cell components with bacteriophages and/orbacteriophage proteins; b) subsequent incubation of the sample,containing the bacterial cells or cell components and the bacteriophagesand/or bacteriophage proteins, with a solid support, wherein the solidsupport exhibits one or more different coupling group(s) on its surfacebinding the bacteria and/or bacteriophage proteins; and c) separatingthe solid support with the bacterial cells or cell components boundthereto via the bacteriophages and/or bacteriophage proteins from thesample.
 2. A method according to claim 1, wherein the coupling group isa lectin, receptor or anticalin.
 3. A method according to claim 1,wherein the coupling group is a streptavidin or Avidin and thebacteriophage proteins are coupled with biotin or a strep-tag.
 4. Amethod according to claim 1, wherein the solid support is a magneticparticle, agarose particle, glass particle, luminex particle, reactiontube, or a microtiter plate.
 5. A method according to claim 1, whereintwo or more different bacteriophages and/or bacteriophage proteins areadded.
 6. A method for the selective purification of bacterial cells orcell components, comprising the following steps: a) contacting a samplecontaining bacterial cells and/or cell components with a magneticparticle, on the surface of which bacteriophages and/or bacteriophageproteins are applied; and b) separation of the magnetic particle withthe bacterial cells and/or cell components bound thereto from thesample.
 7. A magnetic particle coated with bacteriophages and/orbacteriophage proteins.
 8. A magnetic particle according to claim 7,wherein the bacteriophages and/or the bacteriophage proteins areselected from the group consisting of 0c1r, 10tur, L2, L51, M1, MVG51,MV-L1, O1, SpV1, V1, V1, V2, V4, V5, 108/016, 119, 29, 37, 43, 51, 59.1,A1-Dat, Aeh2, Bir, M1, MSP8, ø115-A, ø150A, ø31C, P-a-1, PhiC, R1, R2,SK1, SV2, VP5, Ap3, Ap4, Mm1, Mm3, Mm4, Mm5, phiUW 51, 43, 44RR2.8t, 65,Aeh1, PM1, PIIBNV6, PS8, psi, PT11, 8764, A5/A6, A6, PM2, W11, W2, W4,W7, 1A, alpha, AP50, BLE, F, G, GA-1, II, IPy-1, mor1, MP13, MP15, ø105,ø29 (phi 29), øNS11, PBP1, PBS1, SP10, SP15, SP3, SP8, SPP1, SPβ, SPy-2,SST, type,168, W23, SP50, W23, SP01, MAC-1, MAC-2, MAC-4, MAC-5, MAC-7,Tb, øCb12r, øCb2, øCb23r, øCb4, øCb5, øCb8r, øCb9, øCP18, øCP2, øCr14,øCr28, O11, P13, O2, O3, O5, O6, O8, 1, phiCPG1, Ceβ, F1, HM2, HM3, HM7,7/26, A, A19, AN25S-1, Arp, AS-1, BL3, CONX, MT, N1, øA8010, S-6(L),β,A-4(L), AC-1, LPP-1, S-2L, S-4L, SM-1, P1, T1, TuIa, TuIb, TuII, 1ø3,1ø7, 1ø9, 2D/13, Ae2, alpha10, alpha3, BE/1, BF23, dA, delta1, delta6,dø3, dø4, dø5, Ec9, eta8, f1, fd, G13, G14, G4, G6, HK022, HK97, HR,lambda, M13, M13 mp18, M20, MM, MS2, Mu, O1, ø80, øA, øR, øX174, PA-2,P1, P1D, P2, P22, Qβ, R17, S13, St-1, T1, T2, T3, T4, T5, T6, T7, WA/1,WF/1, WW/1, zeta3, ZG/2, ZJ/2, C21, omega 8, U3, chi, FC3-9, μ2, 01,11F, 121, 1412, 3, 3T+, 50, 5845, 66F, 7480b, 8893, 9, 9266, al,alpha15, b4, B6, B7, Beccles, BZ13, C-1, C16, C2, C-2, DdVI, Esc-7-11,f2, fcan, FI, Folac, fr, GA, H, H-19J, I2-2, 1alpha, ID2, If1, If2, Ike,JP34, JP501, K19, KU1, M, M11, M12, MS2, NL95, ø92, øI, Øii, Omega8,pilHalpha, PR64FS, PRD1, PST, PTB, R, R17, R23, R34, sd, SF, SMB, SMP2,SP, β, ST, tau, tf-1, TH1, TW18, TW28, ViII, VK, W31, X, Y, ZG/1, ZIK/1,ZJ/1, ZL/3, ZS/3, AP3, C3:, 1b6, 223, fri, hv, hw222a, øFSW, PL-1, y5,1, 643, c2, kh, m13, P008, P127, 1358, 1483, 936, 949, BK5-T, c2, KSY1,P001, P008, P107, P335, PO34, PO87, pro2, 4211, psi M2 (ΨM2), N1, N5,Br1, C3, L3, I3, lacticola, Leo, ø17, R1-Myb, N13, N18, N24, N26, N36,N4, N5, X1, X10, X24, X3, X5, X6, D3, D4, 22, 32, AU, C-2, P1, P2, P3,P4, Phi CT, phi CTX, PB-1, 12S, 7s, D3, F116, gh-1, gh-1, Kf1, M6, ø1,øKZ, øW-14, Pf1, W3, 2, 16-2-12, 2, 317, 5, 7-7-7, CM1, CT4, m, NM1,NT2, ø2037/1, ø2042, øgal-1-R, WT1, Mp1, MP2, W1, epsilon15, Felix 01,16-19, 7-11, H-19J, Jersey, N4, SasL1, ViI, ZG/3A, San21, A3, A4, P22,4, C1/TS2, Sp1, 107, 187, 2848A, 3A, 44AHJD, 6, 77, B11-M15, Twort, 182,2BV, A25, A25-24, A25-omega8, A25-PE1, A25-VD13, CP-1, Cvir, H39, P23,P26, phi A.streptomycini III, phi8238, phiC31, S1, S2, S3, S4, S6, S7,SH10, Tb1, Tb2, Ts1, 06N-22P, 06N-58P, 06N-58P, 4996, alpha3alpha, I,II, III, IV, kappa, nt-1, OXN-100P, OXN-52P, v6, Vf12, Vf33, VP1, VP11,VP3, VP5, X29, Cf, Cf1t, RR66, 8/C239, phiYeO3-12, and YerA41.
 9. Amagnetic particle according to claim 7, having a diameter of about 0.5to about 4 μm or about 0.8 to about 1.8 μm.
 10. A bacteriophage proteincomprising a strep-tag or a his-tag.
 11. A bacteriophage proteinaccording to claim 10, whereby the tag exhibits an amino acid sequenceaccording to SEQ ID NO: 5, 6 or
 7. 12. A bacteriophage protein accordingto claim 10, whereby the bacteriophage protein is the p12 protein of thephage T4.
 13. A bacteriophage protein according to claim 10, whereby thebacteriophage protein is produced via DNA recombination technology. 14.A nucleic acid encoding a bacteriophage protein according to claim 10.15. An amino acid segment having a sequence according to SEQ ID NO: 6, 7or
 8. 16. A method for the isolation of nucleic acids or cell componentscomprising contacting a magnetic particle of claim 7 with a biologicalsample.
 17. A kit for the purification of bacterial cells and/or cellcomponents, comprising the magnetic particles according to claim
 7. 18.A kit for the purification of bacterial cells and/or cell components,comprising the bacteriophage proteins according to claim
 10. 19. Themethod of claim 16, wherein said method comprises plasmid preparation.20. The method of claim 16, wherien said cell components compriselipopolysaccharides, endotoxines or exopolysaccharides.
 21. A method forthe isolation of nucleic acids or cell components comprising contactinga bacteriophage protein according to claim 10 with a biological sample.22. The method of claim 21, wherein said method comprises plasmidpreparation.
 23. The method of claim 21, wherien said cell componentscomprise lipopolysaccharides, endotoxines or exopolysaccharides.